1. Field of the Invention
The present invention relates to cyclodextrin glycosyltransferases (CGTases) EC 2.4.1.19 for producing .gamma.-cyclodextrin, to processes for their preparation, and to their use.
2. The Prior Art
As a rule, cyclodextrins are prepared from starch or starch-like substrates. The starch is converted enzymically by CGTases into cyclodextrin (CD). Irrespective of the CGTase used for the reaction, the starch is, for thermodynamic reasons, converted mainly into .beta.-CD if the reaction is carried out until the thermodynamic equilibrium is reached (maximum CD yield). However, in the initial phase, at the beginning of the starch-conversion reaction, the enzymes used for the conversion differ in the composition of the mixture of primary products. A distinction is made between .alpha.-, .beta.- or .gamma.-CGTases in dependence on the main product, .alpha.-, .beta.- or .gamma.-CD, which is formed by the enzyme in this initial phase.
Hitherto, those enzymes which are suitable for the industrial production of CD, and have already been used, have been detected exclusively in bacteria. Hitherto, .alpha.-CGTases have been identified exclusively in Bacillus macerans (J. Bacteriol. (1986) 166, pp. 1118-1122), Bacillus stearotherrnophilus (GB 2169902) and Klebsiella oxytoca (Gene (1986) 47, pp. 269-277). .beta.-CGTases have been detected, for example, in Bacillus circulans (Appl. Microbiol. Biotechnol. (1990) 34, pp. 229-230) , Bacillus megaterium (U.S. Pat. No. 3,812,011) , Bacillus ohbensis (JP 74124285), Microcococcus sp. (EP 0017242) and in alkalophilic bacilli which have not been exactly classified taxonomically, such as Bacillus sp. 38-2 (J. Gen. Microbiol. (1988) 134, pp. 97-105), 17-1 (Proceedings of the 4th International Symposium on Cyclodextrins (1988), pp. 87-92, 1011 (Appl. Microbiol. Biotechnol. (1987) 26, pp. 149-153), and 1-1 (Proceedings of the 4th International Symposium on Cyclodextrins (1988), pp. 71-76). Enzymes having an initially high .gamma.-CD-forming activity have been described in Bacillus subtilis 313 (Agric. Biol. Chem. (1986), 50, pp. 2161-2162), Bacillus sp. A1-6 (J. Ferment. Bioeng. (1990) 70 (3), pp. 150-154), and Bacillus sp. 290-3 (Proceedings of the 4th International Symposium on Cyclodextrins (1988), pp. 87-92).
The three-dimensional structure of the .beta.-CGTase from Bacillus circulans was elucidated by X-ray structural analysis (J. Mol. Biol. (1991) 217, pp. 737-750). It was possible to deduce from this structure which amino acid residues might be able to directly participate in the construction of the substrate binding site and of the active center of this CGTase, but not which amino acid residues determine the product specificity of this CGTase (Biochemistry (1992) 31, pp. 8740-8746).
Since the CGTases used in the industrial preparation of cyclodextrins always afford mixtures composed of several cyclodextrins when converting starch into cyclodextrins, various processes have been developed for isolating pure cyclodextrins (.alpha., .beta. or .gamma.):
defined CD's can be separated chromatographically from the product mixtures, e.g., on the basis of their differences in molecular weight (described, for example, in U.S. Pat. No. 4,808,232); PA1 when converting starch enzymically into cyclodextrins, complex-forming agents are added which only react with one defined CD, thereby, for example, forming an insoluble complex which can be physically separated out of the reaction mixture. Subsequently, the complex is resolved and the homogeneous CD isolated (described, for example, in EP 0291067); PA1 by means of adding an organic solvent, such as, for example, ethanol, to the reaction mixture, the product composition can be shifted in the direction of .gamma.-CD when a .gamma.-CGTase is used (J. Ferm. Bioeng. (1990) 70 (3), pp. 150-154). PA1 Journal of Fermentation and Bioengineering (1990) 70 (3), pp. 190-192: The preparation of .gamma.-CD using the .beta.- and .gamma.-CD-forming CGTase from Bacillus sp. AL-6 in the presence of ethanol, which effects an amplified production of .gamma.-CD. PA1 JP 87 25,976: The preparation of .gamma.-CD using the .gamma.-CGTase from Bacillus sp. 313. PA1 - EP 291,067: Preparation of 7-CD using the CGTase from Bacillus macerans. The product specificity for .gamma.-CD is achieved by adding a complex-forming agent, e.g. cyclohexadec-8-en-1-one. PA1 - DE 40 09 822: Production of 7-CD using the .gamma.-CGTase from Bacillus sp. 290-3.
In each of the processes, those CGTases are optimally used which have as high an initial product-formation preference as possible for the CD which is to be prepared in pure form.
The specificity of the .beta.- and .gamma.-CGTases known hitherto is adequate for the industrial production of the corresponding cyclodextrins. By contrast, none of the known .gamma.-CGTases possesses product specificity permitting a comparable production of .gamma.-CD.
In order to prepare .gamma.-CD, therefore, it was proposed in JP 03053892 to convert .alpha.- and/or .gamma.-cyclodextrins enzymically into .gamma.-CD by adding the .gamma.-CD-specific complex-forming agent glycosylglycyrrhizin, maltose and a CGTase.